Characterization of Specific RAPD Markers of Virulence in Trichomonas vaginalis Isolates.

BACKGROUND
As for human trichomoniasis the host-parasite relationship is very complex, and the broad ranges of clinical symptoms are unlikely be attributable to a single pathogenic mechanism. Specific Random Amplified Polymorphic DNA (RAPD) markers of 490 bp, 720 bp and 460 bp using the primers Tv-5, OPA-6 and OPA-11, respectively, were reported. This was the first description of possible genetic virulence markers of the infection by T. vaginalis. The aim of this study was to characterize the specific RAPD markers in order to elucidate their importance on virulence of this illness.


METHODS
The selected specific RAPD fragments were cloned and sequenced. The obtained sequences were compared by the BLAST algorithm.


RESULTS
The nucleotide sequence of the Tv-5490 RAPD marker exhibited significant similarity to T. vaginalis hypothetical G3 leucine rich repeat (LRR) family protein (e-value: 6e-14) and Giardia lamblia leucine rich repeat protein 1 virus receptor protein (e-value: 6e-14 and 2e-12) ; however, the OPA-6720 and OPA-11460 showed no significant similarity with any coding published sequence. All the evaluated strains showed the presence of the LRR gene.


CONCLUSION
These results demonstrate a possible role of this gene in the virulence of T. vaginalis and in the parasite infection with Trichomonas virus as a possible virus receptor. Further analysis of this gene and encoded protein will allow determining the role that they play in the isolates virus susceptible or resistant phenotypes.


Introduction
richomonas vaginalis is a parasitic protozoan that is the cause of trichomoniasis, a sexually transmitted disease of worldwide importance. Annual incidence of trichomoniasis is more than 276.4 million cases worldwide (1). Both sexes can be affected by the disease. It can demonstrate a wide clinical forms variation in symptoms, from an asymptomatic presentation to severe sequelae. For women, whom are primarily affected in the vulva, vagina, and uterine cervix, and secondarily, in the urinary tract, trichomoniasis can have major health consequences. In addition, infection with T. vaginalis in pregnant women has been associated with premature rupture of membranes and premature delivery (2). Trichomoniasis has also been associated with an increased risk of human immunodeficiency virus (HIV) acquisition and transmission and cervical cancer (3)(4)(5).
The host-parasite relationship is very complex, and the broad range of clinical symptoms are unlikely be attributable to a single pathogenic mechanism. The exact mechanisms of pathogenesis resulting in the wide variation in clinical presentation (from asymptomatic to severe symptomatic) has not been clearly elucidated to date (6)(7).
In population biology, both direct and nondirect techniques are used for the identification of the genetic source of variation and the genetic locus of disease or quality strain. The non-directed approaches used random genome scanning methods initially to generated polymorphic genetic map markers, which then can be linked to traits of interest. The conversion of mapped random markers into sequence-characterized loci necessitates the isolation of the marker DNA fragment and the determination of its DNA sequence (8).
RAPD markers are a non-directed approach in which polymorphic PCR products are amplified from genomic templates using monomer oligonucleotide primers, which generally anneals with multiple sites in different regions of the genome and amplifies several genetic loci simultaneously. Polymorphism of amplified fragment are caused by: 1) base substitution or deletion in the priming site, 2) insertion that render priming site too distant to support amplification or 3) insertion or deletion that change the size of the amplified fragment (9). In addition, RAPD markers have been demonstrated as useful genetic marker for a variety of eukaryotic organism (10). It is desirable to develop RAPD methodology because of its versatility, as the ready detection of informative loci in all species underpins the universality of the technology.
The genetic polymorphism using the RAPD technique has been correlated with different isolates biological properties such as metronidazol susceptibility (11)(12)(13)(14)(15), the presence of Mycoplasma in strains of T. vaginalis (13,15), the presence of T. vaginalis virus (TVVs) infections (11)(12)(13)16) and the clinical manifestations in patients (11,14,17,18). Meade et al. (19) reported the genetic diversity in T. vaginalis isolates using the RFLP of cytoplasmic hsp70 gene and proposed that parasite isolates are organized into two clonal linages, however the authors did not correlated the phylogenetic relationships with clinical presentation.
The association between the genetic polymorphism and clinical presentation in patients (symptomatic and asymptomatic) found by different authors (11,14,17,18) demonstrate the role of the parasite in the virulence.
In a previous paper, our group found a specific RAPD marker of 490 bp in all symptomatic isolates but not in the asymptomatic ones using the primer Tv-5 (14). When using another group of primers (OPA-1 to OPA-20) we find other two specific RAPD markers of 720 bp and 460 bp using the primers OPA-6 and OPA-11 (18). This was the first description of possible genetic markers of the virulence of the infection by T. vaginalis.
The aim of this study was to characterize the specific virulence RAPD markers in order to elucidate their possible biological function.

Trichomonas vaginalis isolates
Thirty-seven fresh T. vaginalis isolates, collected from sexually active adolescent women consulting by sexually transmitted infections in gynecobstetrics hospitals ''Ramón González Coro" and ''Eusebio Hernández" in Havana City, Cuba, during 1999 to June 2000, were enrolled in the study.
Ethical approval of the study had been obtained from the Institute of Tropical Medicine Pedro Kouri and Gynecobstetric Hospitals Ethics Committee. Patients were informed of the about the study and following written informed consents from them or their parents, and clinical data were collected.
Various laboratory tests were carried out, to determine if other sexually transmitted microorganisms, such as yeast infections, bacterial vaginosis, gonorrhoea, Chlamydia trachomatis, Mycoplasma hominis, human papilomavirus and HIV were present in patients (18,20). The studied isolates are free of these other concomitant microorganisms. All these strains are deposited in the culture collection of the Parasitology Department from the Institute of Tropical Medicine ''Pedro Kouri", Havana, Cuba. All Trichomonas isolates were maintained as axenic cultures cultivated at 37 °C in TYI-S-33 Diamond's medium (21), supplemented with 10 % heat inactivated calf serum. In the exponential growth phase parasites (2 x 10 6 ) were placed in -70 °C freezer for the later evaluation of PCR gene detection.
Medical doctors recorded symptoms (vaginal discharge, pruritus, dysuria, and dyspareunia among others) and signs (vaginal discharge, cervical, vaginal or vulvar erythema) by formal questionnaires and clinical examinations of patients. According to clinical presentation, the isolates were classified as asymptomatic (no symptoms and signs) or symptomatic (with one or more symptoms and signs). In addition, these isolates are well characterize genetically by RAPD and the infection with Trichomonas virus has been reported (16, 18, 22) ( Fig. 1).

RAPD virulence markers
Three RAPD genetic markers associated to virulence were obtained using the RAPD technique during the genetic polymorphism analysis of T. vaginalis isolates (14,18). The primers Tv-5, OPA-6 and OPA-11 amplified a single, bright band of approximately 490 bp, 720 bp and 460 bp in all symptomatic isolates and was absent in the asymptomatic isolates (Fig. 1). These bands were named Tv-5 490, OPA-6 720 y OPA-11 460 . The putative virulent RAPD marker was successfully cloned and sequenced from six T. vaginalis isolates (A170, C334, A42, C187, C173, C351), choosing two isolates representative of each one of the three main RAPD genetic groups (II a, b and c) and infected with Trichomonas virus (Fig. 1) (16,18,22).

Cloning and sequencing RAPD virulence markers
The RAPD-PCR fragments were excised from agarose gels with a sterile gel slicer, then extracted and purified by using QIAquick Gel Extraction Kit (Qiagen). The PCR fragments were used as a template for re-amplification with TV-5, OPA-6 and OPA-11 primers for confirmation of the right fragment. The purified PCR fragments were then directly cloned into pGEM-T Easy vector (Promega Madison) following the supplier instruction. The recombinant DNA was used to transform E. coli JM109 competent cells. Recombinants were identified as white colonies on LB plates with X-gal and IPTG. These colonies were further screened by taking stab of a transformant colony, boiled in 20 µl of sterile water and then 1.2 µl of the solution is used in an amplification reaction with the original primer. Plasmid DNA was isolated from putative recombinants with the QIAprep Spin Plasmid Kit (QIAgen) using the standard protocol provided by manufacturer. An aliquot of purified plasmid DNA was analyzed again by PCR, while another aliquot was digested by SalI and NcoI restriction enzyme (Fermentas, Belgium). Sequences of the plasmid DNA were generated using the T7 and SP6 primers for se-quencing both strand of the entire amplicon with dideoxy nucleotide chemistry with the ABIPRISM BigDyeTM Terminator cycle sequencing kit (PerkinElmer, Foster City, CA, USA), and analyzed on an ABI 3730 automated sequencer (PerkinElmer). The consensus sequences of each RAPD markers were obtained using the software package MEGA (23) (Molecular Evolutionary Genetic Analysis Version 4, www.megasoftware.net).

RAPD virulence marker sequences analysis
The sequence data were compared with other nucleotide sequences available throughout the National Center for Biotechnology Information (NCBI, USA) databases using BLASTn and BLASTx programs (Basic Local Alignment Search Tool; 24) algorithm at http://www.ncbi.nlm.nih.gov/BLAST/ of the National Center for Biotechnology Information (NCBI). Also the sequences were compared with the complete genome of T. vaginalis (G3 strain American Type Culture Collection PARA-98, Human, 1973, New England) using the T. vaginalis genome database (TrichoDB, http://trichodb.org/trichodb) Sequence alignments were considered biologically significant when presenting E-values below 10 -10 and 10 -2 for nucleotide and protein sequence, respectively (25). The ORF signatures were detected using the NCBI ORF finder tool.

Nucleic acid extraction
The T. vaginalis pellet from each isolate was used for the extraction of nucleic acids (22). Genomic DNA was used as template for the detection of T. vaginalis Leucine Rich Repeat (LRR) family protein gene using specific PCR technique.

PCR amplification of Leucine Rich Repeat (LRR) family protein gene in T. vaginalis isolates
PCR primers were designed in order to amplify the LRR family protein gene of T. vaginalis based on a sequence of a G3 strain reported in a GenBank with the accession number XM_001298269. Two primers, named LRRTv_F (5'-GCGACTGTGCTTATCAC-GAAATAG-3') and LRRTv_R (5'-GTTTAGGG-CGGAGTATGTTGC-3'), were designed to amplify a fragment of around 2235 bp using the program Primer3 (http://simgene.com/Primer3). The primer sequences were checked with the Oligo Properties Calculator

Results
By aligning all clone sequences, a consensus sequence was generated which actually consisted of 466 pb, 700 pb and 440 pb (Gen-Bank accession No. JQ699181, JQ699182, JQ699183). The closest similarities of RAPD virulence marker sequences are shown in Table 1. The sequence of the Tv-5 490 RAPD marker exhibited significant sequence similarity to T. vaginalis hypothetical G3 LRR family protein and G. lamblia LRR protein 1 virus receptor protein. The sequence of each one of Tv-5 490 RAPD marker corresponding to each isolates chosen were deposit in the GenBank with the accession numbers KF318415-KF318420.
However, BLAST results revealed that OPA-6 720 and OPA-11 460 did not have significant similarity with any known coding nucleotide sequences. The consensus sequences of these markers correspond to non-coding region of T. vaginalis genome.
A PCR was designed in order to amplify the gene to codify the LRR family protein in T. vaginalis. The expected PCR product was amplified in all of isolated evaluated (Fig. 1B).

Discussion
The leucine rich repeat family protein constitute a group of proteins included in a large BspA like proteins, one of the three mains surface proteins family (BspA like proteins, GP63-like proteins and adhesion or others) (26,27). The BspA-like proteins (TvBspA) are the largest gene family encoding potential surface proteins and share a specific type of LRR, named TpLRR after a membrane protein from Treponema pallidium (28), shared with T. forsythensis BspA and Treponema denticola LrrA proteins (29). LRR is a versatile binding motif found in a variety of proteins and is involved in protein-protein interaction (30). The TvB-spA proteins could represent receptors mediating endocytosis of various host proteins with by their TpLRR likely involved in binding to ligands (31). T. vaginalis has over 300 candidate surface proteins, which belong to ten different families with at least one inferred transmembrane domain and share one or more features with other pathogens surface proteins (32).
The RAPD Tv-5 490 marker also matches homology to G. lamblia LRR protein 1, virus receptor protein. In G. lamblia, one dsRNA virus has been identified, as Giardiavirus (33). The time course of Giardia virus infection in the parasite reveals that the virus enters susceptible parasite cells via endocytosis, in which the lysosomal-like peripheral vacuoles serve a translocation ports for the virus to reach the cytoplasm (34). Sepp et al. (35) proposed that the Giardia virus invasion is most likely mediated by a receptor-directed endocytosis in G. lamblia. This putative receptor(s) is expected to be present on the surface of the virus susceptible cells, responsible for the attachment and internalization of the Giardia virus, but missing from the resistance cells.
T. vaginalis can be infected with double stranded RNA (dsRNA) viruses known as T. vaginalis virus (TVVs). This virus infection induces various phenotypic changes that may affect T. vaginalis virulence (36,37). In previous paper, the presence of TVVs in a group of clinical isolates was detected (22). A significant association between TVVs detection and the presence of particular clinical symptoms and signs in infected patients was demonstrated. This evidence constitutes an indicative of a possible clinical significance of TVV infection in T. vaginalis isolates (20). More recently, we found an association between the TVV infection and cytoadhesion level of isolates (16), as another evidence of a possible role of the virus in the virulence of human trichomoniasis. The results described by Ficharova et al. (38) support the novel concept that protozoan endobiont viruses and viral dsRNA sensors of the human host represent a critical target to exacerbate the disease through signaling of immunoinflammatory responses by human epithelial cells. More recently, Parent et al. (39) provide both mechanistic and translational insights concerning the role of Trichomonas viruses in aggravating diseases attributable to T. vaginalis. All these results demonstrate the role of the virus in the virulence of the parasite.
The analysis of polymorphism among 37 isolates by RAPD showed significant association with the presence of TVV, demonstrating the existence of concordance between the genetic relatedness and the presence of TVV in T. vaginalis isolates. This result could point to a predisposition of T. vaginalis for the viral enters and/or survival (18). In this paper, after the characterization of RAPD markers associated to the virulence, the presence of a gene that it could be involved in the entrance of the TVVs, as it happens in G. lamblia with Giardia virus is revealed (35). Today, it is not known how the virus enters to the protozoan and whether the virus was acquired from the host in which protozoan resided.

Conclusion
Our results demonstrate a possible role of the parasite in the infection with TVVs. All of studied isolates, susceptible or resistant to TVVs, presents the LRR family protein gene. Further studies focused in the polymorphism characterization of the gene and it coding protein are needed, in order to determine the role of this gene in the internalization of TVVs to the protozoan. In a future, the Tv-5 490 marker will be used to converting the RAPD marker into a single locus PCR-based marker of a sequence-characterized amplified region (SCAR) with higher sensitivity and reproducibility.